The planetary biology of cytochrome P450 aromatases

BMC Biology The planetary biology of cytochrome P450 aromatases Eric A Gaucher 2 Logan G Graddy 1 Tang Li 2 Rosalia CM Simmen 0 Frank A Simmen 0 David R Schreiber 2 David A Liberles 5 Christine M Janis 4 Steven A Benner 3 0 Department of Physiology & Biophysics, Medical Sciences & Children's Nutrition Center, University of Arkansas , 1120 Marshall Street, Little Rock AR, 72202 , USA 1 Department of Psychiatry, Duke University Medical Center , Durham, NC 27708 , USA 2 Foundation for Applied Molecular Evolution , 1115 NW 4th Street, Gainesville FL 32601-4256 , USA 3 Department of Chemistry, University of Florida , Gainesville FL 32611-7200 , USA 4 Ecology and Evolutionary Biology, Brown University , Providence RI 02912 , USA 5 Computational Biology Unit, Bergen Center for Computational Science, University of Bergen , 5020 Bergen , Norway Background: Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system. Results: Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases-enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene. Conclusions: This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems. - Background The emergence of complete genomes for many organisms, including humans, has created the need for hypotheses concerning the "function" of specific genes that encode specific proteins. While "function" is interpreted by different workers in different ways [1], Darwinian theory (by axiom) requires that the term be connected to fitness; natural selection is the only mechanism admitted by theory to generate functional behavior in a living system, macro or molecular. This, in turn, implies that the hypotheses about function have a "systems" component, including the interaction of the protein with other proteins, their impact on the physiology (defined broadly) of the cell and organism, and the consequences of physiology in a changing ecosystem in a planetary context [2]. Systems hypotheses can be supported by information from many areas. Geology, paleontology, and genomics, for example, provide three records that capture the natural history of past life on Earth. At the same time, structural biology, genetics, and organic chemistry describe the structures, behaviors and reactivities of proteins that allow them to support present life. It has been appreciated that a combination of these six types of analysis provides insights into functional behavior of proteins that cannot be provided by any of these alone [2]. Over the long term, we expect that the histories of the geosphere, the biosphere, and the genosphere will converge to give a coherent picture showing the relationship between life and the planet that supports it. This picture will be based, however, on individual cases that serve as paradigms for making the connection. The aromatase family of proteins offers an interesting system to illustrate the power of this combination as a way to create hypotheses regarding protein function within a system [3]. These hypotheses are not "proof", of course, but are limiting in genomics-inspired biological experimentation, now that genomic data themselves are so abundant. Aromatases are cytochrome P450-dependent enzymes that use dioxygen to catalyze a multistep transformation of an androgenic steroid (such as testosterone) to an estrogenic steroid (such as estradiol) (Figure 1). The protein plays a key role in normal vertebrate reproductive biologya role that appears to have arisen before fish and tetrapods (land vertebrates, including mammals) diverged some 375 million years ago [4]. Aromatase is important in modern medicine as well, especially in breast and other hormone-dependent cancers [5]. Different numbers of aromatase genes are found in different vertebrates. Two aromatase genes are known in teleost fish [6,7]. Only a single gene is known in the horse [8], rat [9], and mouse [10]. Cattle have both a functional gene and a pseudogene built from homologs of exons 2, 3, 5, 8, and 9 of their functional gene; these are interspersed with a bovine repeat element [11,12]. In several mammalian species, including humans and rabbits, a single gene yields multiple forms of the mRNA for aromatase in different tissues via alternative splicing [13-16]. A still different phenomenology is observed in the pig (Sus scrofa). Three different mRNA molecules had been reported in different tissues from pig [17-21]. Compelling evidence then emerged that the three variants of mRNA identified in cDNA studies arose from three paralogous genes [22], rather than from a single gene differentially spliced [23]. This implies that the three aromatase paralogs in pigs arose via gene duplications relatively recent in geologic time. Hypotheses relating to the function of the three aromatase paralogs depend in part on when those duplications took place. If they were very recent, the three genes might have helped pigs adapt to domestication. If they pre-dated the divergence of pigs and fish [6], they may have different roles that are very fundamental to reproductive endocrinology in vertebrates. We apply here a series of tools to generate better hypotheses concerning the aromatase family of paralogs in swine. Resul (...truncated)